EXPRESSION

SYSTEM:
SACCHAROMYCES
CEREVISAE &
PISCIA PASTORIS

SUBMITTED TO:

DR. BINDU
.

– Expression systems : these are

genetic constructs that are designed
to produce a protein or an RNA,
either inside or outside the cell.
– Expression vectors or expression
construct : is usually a plasmid or
virus designed for protein expression
in cells.
Expression system in
saccharomyces cerevisiae
•Fungus share many of the molecular ,
genetic and biochemical features of other
higher eukaryotes and are therefore a good
choice for heterologous protein production
. – ADVANTAGES:
– It is easy and cheap to grow on simple media in small vessels and
large scale bioreactor
– Tremendous range of vectors and genetic resources available ,
including promoters and regulatory systems
– It shows no health hazards
– At present more than 50% of world supply of insulin is produced by
s. cerevisiae
– S.cerevisiae is capable of carrying out many post translational
modifications .
– Yeast normally secrets few proteins that , when it is engineered for
extracellular release of recombinant proteins
S . cerevisiae vectors
– Three main classes of s.cerevisiae vectors :
➢Episomal , or plasmid vector (yeast episomal plasmid
{YEps} )
➢Integrating vectors (yeast integrating plasmids {Yips})
➢Yeast artificial chromosome (YAC)
Yeast episomal plasmids
– is a small circular plasmid found in most
natural strains of S. cerevisiae.
– Its is based on a high copy number 2µm
plasmid .
– The vector replicates independently of
host chromosomes via a single origin
maintained in more than 30
copies per cell
– It is a shuttle vector , which means it can
replicate and exist in both a bacterial and
yeast system .
Yeast integrated plasmids
– These vectors are the vectors that do not
replicate autonomously, but integrate into the
genome at low frequencies by homologous
recombination
– The size of vector is 5.5 kb.
– The copy number is only 1 ,
– Transformation efficiency of vector is less than
1000 cells per microgram
– As YEps are unstable under conditions of large
scale growth a hetrologus gene is integrated
into host genome to provide more reliable
production system
Yeast artificial chromosome
– It is designed to clone a large segment of
DNA(100 kb), which is then maintained as a
separate chromosomes in the host yeast cells
– YAC system is highly stable and has been used
for physical mapping of human genomic DNA,
the analysis of large transcription unit.
– A YAC vector mimic a chromosome because it
has a sequence that acts as a origin of DNA
replication
– Few contain selectable marker gene that are
independent of cloning site
 Protein production steps
–
Gene expression in Post translational Protein secretion and Downstream
new host modifications intracellular transport processing

Regulation of gene Translocation to The secretory pathway: Chemical character

Expression secretory pathway ER » Golgi » secretory of protein
Molecular biology Folding and modifications vesicle » PM Protein engineering
Methods Glycosylation Recovery and
purification
Protein production
– Proteins may be intracellular or extracellular
– Intracellular proteins may be produced intracellularly or directed out of cell
by adding a signal sequence or by construction of a fusion protein with an
extracellular protein
– In bacteria proteins may end in periplasmic space leads to formation of
inclusion bodies.
– Production may be enhanced by use of fusion protein strategies
– PCR has made it much easier to make the constructions needed
.– Homologous production :production by gene donating host organism
– Heterologous production : production by a different host organism
– Classical mutagenesis can be used for increasing homologous expression
– Recombinant production may be facilitated by introducting the gene in an
autonomously replicating plasmids or by integrating it to genome
– Copy number of genes in organisms can be increased.
– The degradation of product may be controlled by e.g. use of protease
defective mutants or by changing the sequence of protein .
 YAC cloning system
– A YAC is designed to clone a large segment of DNA (100 kb) , which is then maintained as a seprate
chromosome in host yeast cell .
– It is highly stable and has been used for physical mapping of human genomic DNA, the analysis of
transcription units, and genomic libraries .
– It has a sequence that act as ARS for replication , centromere for cell division , and telomere for stability .
– To date , they have not been used as expression system for the commercial production .
YEAST CLONING SYSTEM
Intracellular production

– Example human enzyme :super oxidase dismutase (sod)

– Superoxide anion is a byproduct of oxygen utilization in aerobic organisms
– In humans it stimulates inflamatory response of phagocyts and direct
leukocytes to the site of infection
– It is also produced when blood is allowed to reenter an organ after a surgical
procedure .
.– to much of this molecule and its derivative can
cause cellular damage and to minimize the
potentially cytotoxic effects
– Naturally occurring Cytoplasmic enzyme (Cu/Zn-
SOD) and superoxide radical gives hydrogen
peroxide in presence of hydrogen ions which further
gives water and oxygen in presence of
catalase/peroxidase.
– To prevent superoxide aion damage during
reperfusion Cu/Zn- SOD can be used .
– In addition Cu/Zn- SOD might act as therapeutic
agent against inflammatory diseases
.
– Initially cDNA was cloned in E. coli expression system
– E. coli host cells remove initiator N-terminal methionine from Cu/Zn SOD protein, but
next amino acid was not catalyzed .
– To produce fully functional protein , the human Cu/Zn SOD cDNA was cloned into Yep
vector
– A leucine-defective (LEU2)yeast strain was transformed with vector, and the cells
were plated on to medium that lacked leucine ,cells with the functional LEU2 gene
,could grow under these conditions .
– The yeast cells produced high level of intracellular Cu/Zn- SOD anacetylated N-
terminal alanine residue.
Secretion of heterologous proteins
– Protein may also be produced for secretion
– In this system , any glycosylated protein is secreted (O or N linked ).
– The coding sequences of recombinant proteins must be cloned downstream of
a leader sequence , the yeast matting type factor α – factor.
– Under these conditions , correct disulfide bond formation , proteolytic
removal of the leader sequence , an appropriate post translational
modifications occur , and an active recombinant protein is secreted .
– The leader peptide is removed by endoprotease that recognize the lys-Arg.
– For example , a properly processed an active form of protein hirudin , a
powerful anticoagulant protein cloned from a leech , was synthesized and
secreted by an S.cerevisiae
. – A YEp vector that had the prepro-α-factor sequence added to the hirudin coding
sequence to allow expression that is cleaved away in processing
– Leaves active hirudin which is secreted.
– Producing a recombinant protein for use in human therapeutics in yeast rather than
in bacteria is to ensure the proper folding.
 Disadvantages of S. cerevisiae
– Though S. cerevisiae is successfully used to produce recombinant proteins for human,
it has major drawbacks.
– The level of proteins production is low.
– There is the tendency for hyper glycosylation resulting in change of protein function.
– Proteins are often retained in periplasm, increasing time and cost for purification.
– It produces ethanol at high cell densities, which is toxic to cells.
Pichia pastoris expression system
– P.pastoris , a second specis of yeast ia able to synthesize large amount of
recombinant protein and its glycosylation abilities a very similar to those of
animal cells .
– It is methylotropic yeast that is able to uthilice methanol as a source of
carbon and energy .
– Glycosylation occurs to lower extent and the linkages between sugar
residues are of the α-1,2 type .
– It does not produce ethanol
– It normally secrets very few protein, thus simplifying the purification of
secreted recombinant proteins .
. – Expression vectors for P. pastoris make use of alcohol oxidase (AOX) promoter, which
is induced by methanol.
– A double recombination event between AOX1p and AOX1 regions of vector and
homologous segment of chromosome DNA results in insertion of the DNA carrying
the gene of interest and the HIS4 gene.
 INTEGRATION OF DNA INTO SPECIFIC P. PASTORIS
CHROMOSOME SITE BY SINGLE OR DOUBLE RECOMBINATION